CN111505171B - Method for identifying three medicinal polygonatum varieties - Google Patents

Method for identifying three medicinal polygonatum varieties Download PDF

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CN111505171B
CN111505171B CN202010512201.5A CN202010512201A CN111505171B CN 111505171 B CN111505171 B CN 111505171B CN 202010512201 A CN202010512201 A CN 202010512201A CN 111505171 B CN111505171 B CN 111505171B
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polygonatum
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CN111505171A (en
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蔡永萍
冉锐
童金凤
张文武
金青
程曦
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Anhui Agricultural University AHAU
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    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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Abstract

The invention provides a method for identifying three medicinal polygonatum varieties, belonging to the technical field of quality control of traditional Chinese medicines. The method comprises the steps of extracting and derivatizing three medicinal polygonatum sibiricum, analyzing and detecting metabolites in the medicinal polygonatum sibiricum by using a gas chromatography-mass spectrometry, and screening differential metabolites of the three medicinal polygonatum sibiricum by using a multivariate statistical method, wherein the differential metabolites are used as characteristic metabolites for identifying three medicinal polygonatum sibiricum varieties, so that the identification of the three medicinal polygonatum sibiricum varieties is realized. The method provided by the invention has high accuracy and reliability and high reliability of experimental results, provides scientific basis for quality identification and quality control of the three medicinal polygonatum kingianum, and is beneficial to quickly and accurately identifying the authenticity and quality of the medicinal polygonatum kingianum sold in the market.

Description

Method for identifying three medicinal polygonatum varieties
Technical Field
The invention relates to the technical field of quality control of traditional Chinese medicines, in particular to a method for identifying three medicinal polygonatum varieties.
Background
The medicinal rhizoma Polygonati is dried rhizome of Polygonatum sibiricum Red, Polygonatum kingianum Coll et Hemsl, and Polygonatum cyrtonema Hua of Polygonatum of Liliaceae, is rich in medicinal active ingredients such as polysaccharide and steroid saponin, and has effects of invigorating qi, nourishing yin, invigorating spleen, moistening lung, and invigorating kidney. However, the medicinal polygonatum rhizome tubers have high similarity, are difficult to identify in appearance, are good in quality and are in a false or true state. Therefore, there is a need to provide an effective analysis method for quality identification and quality control of medicinal polygonatum sibiricum.
Disclosure of Invention
The method provided by the invention has high accuracy and reliability and high reliability of experimental results, and provides scientific basis for quality identification and quality control of the three medicinal polygonatum kingianum.
In order to achieve the above object, the present invention provides the following technical solutions:
a method for identifying three varieties of medicinal polygonatum sibiricum, including polygonatum sibiricum, polygonatum kingianum and polygonatum cyrtonema, comprises the following steps:
(1) grinding the rhizoma polygonati sample, the polygonatum kingianum sample and the polygonatum cyrtonema sample respectively and dispersing the grinded samples in methanol to obtain a methanol dispersion liquid of the rhizoma polygonati sample, a methanol dispersion liquid of the polygonatum kingianum sample and a methanol dispersion liquid of the polygonatum cyrtonema sample;
(2) mixing the methanol dispersion liquid of the polygonatum sibiricum sample, the methanol dispersion liquid of the polygonatum kingianum sample and the methanol dispersion liquid of the polygonatum cyrtonema sample with a methanol solution of catechol respectively, then carrying out ultrasonic treatment and first solid-liquid separation in sequence, extracting the obtained first liquid materials in a chloroform-water mixed solvent respectively, and then carrying out second solid-liquid separation to obtain second liquid materials which are an extracting solution of the polygonatum kingianum sample, an extracting solution of the polygonatum kingianum sample and an extracting solution of the polygonatum cyrtonema sample respectively;
(3) removing the solvent in the rhizoma polygonati sample extracting solution, the polygonatum kingianum sample extracting solution and the polygonatum cyrtonema sample extracting solution, mixing the obtained mixture with a pyridine solution of hydroxylamine hydrochloride respectively to perform first derivatization treatment, then mixing the obtained mixture with a derivatization reagent to perform second derivatization treatment, and performing third solid-liquid separation to obtain third liquid materials, namely a rhizoma polygonati sample deriving solution, a polygonatum kingianum sample deriving solution and a polygonatum cyrtonema sample deriving solution; wherein the derivatizing agents comprise N, O-bis (trimethylsilyl) trifluoroacetamide and trimethylchlorosilane;
(4) and performing gas chromatography-mass spectrometry analysis by taking the derived liquid of the polygonatum kingianum sample, the derived liquid of the polygonatum kingianum sample and the derived liquid of the polygonatum cyrtonema sample as samples to be detected, performing multivariate statistical analysis on the obtained data, and identifying three medicinal polygonatum kingianum varieties according to the obtained characteristic metabolites.
Preferably, the grinding mode is liquid nitrogen grinding; and (4) sieving the powder obtained after grinding through a 80-mesh sieve, and taking undersize products for later use.
Preferably, the dosage ratio of the rhizoma polygonati to the methanol for the traditional Chinese medicine is 100 mg: 1.4 mL; the concentration of catechol in the methanol solution of catechol is 0.2 mg/mL; the dosage ratio of the rhizoma polygonati sample to the methanol solution of catechol in the methanol dispersion liquid of the rhizoma polygonati sample is 100 mg: 60 mu L of the solution;
the volume ratio of the first liquid material to chloroform and water in the chloroform-water mixed solvent is 1.4 mL: 750 μ L: 1.4 mL.
Preferably, the temperature of the ultrasound is 40 ℃ and the time is 30 min; the extraction method is vortex oscillation for 1 min.
Preferably, the concentration of hydroxylamine hydrochloride in the pyridine solution of hydroxylamine hydrochloride is 20mg/mL, and the volume ratio of the pyridine solution of hydroxylamine hydrochloride to the extract solution of the rhizoma polygonati sample is 60 μ L: 1 mL.
Preferably, the temperature of the first derivatization treatment is 60 ℃ and the time is 120 min.
Preferably, the mass ratio of the N, O-bis (trimethylsilyl) trifluoroacetamide to the trimethylchlorosilane in the derivatization reagent is 99:1, and the volume ratio of the derivatization reagent to the extract solution of the rhizoma polygonati sample is 60 muL: 1 mL.
Preferably, the temperature of the second derivatization treatment is 60 ℃ and the time is 90 min.
Preferably, the operating conditions of the gas chromatograph include, when performing the gas chromatograph-mass spectrometer analysis:
DB-5 quartz capillary column 60m × 250 μm × 0.25 μm; split-flow sample injection with the sample injection amount of 1 mu L and the split-flow ratio of 10: 1; the temperature of a sample inlet is 280 ℃; the ion source temperature is 250 ℃; the interface temperature is 250 ℃; temperature rising procedure: the initial temperature is 40 ℃, the temperature is kept for 5min, the temperature is increased to 280 ℃ at the speed of 8 ℃/min, and the temperature is kept for 5 min; the carrier gas is helium, and the flow rate of the carrier gas is 1 mL/min; the solvent delay time is 14 min; the mass detection range is 33-600 in terms of m/z;
the operating conditions of the mass spectrometer include:
an electrospray ionization source, wherein the temperature of an ion source is 230 ℃, the temperature of a quadrupole rod is 150 ℃, the full scanning mode is adopted, and the electron energy is 70 eV; the scanning range of the quadrupole rods is 35-780 in terms of m/z.
Preferably, the multivariate statistical analysis comprises principal component analysis and orthogonal partial least squares discriminant analysis.
The invention provides a method for identifying three varieties of medicinal polygonatum sibiricum, which comprises the steps of extracting and derivatizing the three medicinal polygonatum sibiricum, analyzing and detecting metabolites in the medicinal polygonatum sibiricum by using a gas chromatography-mass spectrometry, and screening differential metabolites of the three medicinal polygonatum sibiricum by using a multivariate statistical method, wherein the differential metabolites are used as characteristic metabolites for identifying the three varieties of medicinal polygonatum sibiricum, so that the identification of the three varieties of medicinal polygonatum sibiricum is realized. The method provided by the invention has high accuracy and reliability and high reliability of experimental results, provides scientific basis for quality identification and quality control of the three medicinal polygonatum kingianum, and is beneficial to quickly and accurately identifying the authenticity and quality of the medicinal polygonatum kingianum sold in the market.
Drawings
FIG. 1 is a TIC spectrum of three samples of rhizoma Polygonati in example 1;
FIG. 2 is a graph of cluster analysis of three samples of medicinal rhizoma Polygonati in example 1;
FIG. 3 is a graph of PCA scores of three samples of medicinal Polygonatum sibiricum Red in example 1;
FIG. 4 is the S-plot of Polygonatum kingianum and Polygonatum kingianum OPLS-DA in example 1;
FIG. 5 is an S-plot of Polygonatum sibiricum and Polygonatum cyrtonema OPLS-DA in example 1;
FIG. 6 is the S-plot of Polygonatum kingianum and Polygonatum cyrtonema OPLS-DA in example 1.
Detailed Description
The invention provides a method for identifying three medicinal polygonatum kingianum varieties, wherein the three medicinal polygonatum kingianum are polygonatum kingianum, polygonatum kingianum and polygonatum cyrtonema, and the method comprises the following steps:
(1) grinding the rhizoma polygonati sample, the polygonatum kingianum sample and the polygonatum cyrtonema sample respectively and dispersing the grinded samples in methanol to obtain a methanol dispersion liquid of the rhizoma polygonati sample, a methanol dispersion liquid of the polygonatum kingianum sample and a methanol dispersion liquid of the polygonatum cyrtonema sample;
(2) mixing the methanol dispersion liquid of the polygonatum sibiricum sample, the methanol dispersion liquid of the polygonatum kingianum sample and the methanol dispersion liquid of the polygonatum cyrtonema sample with a methanol solution of catechol respectively, then carrying out ultrasonic treatment and first solid-liquid separation in sequence, extracting the obtained first liquid materials in a chloroform-water mixed solvent respectively, and then carrying out second solid-liquid separation to obtain second liquid materials which are an extracting solution of the polygonatum kingianum sample, an extracting solution of the polygonatum kingianum sample and an extracting solution of the polygonatum cyrtonema sample respectively;
(3) removing the solvent in the rhizoma polygonati sample extracting solution, the polygonatum kingianum sample extracting solution and the polygonatum cyrtonema sample extracting solution, mixing the obtained mixture with a pyridine solution of hydroxylamine hydrochloride respectively to perform first derivatization treatment, then mixing the obtained mixture with a derivatization reagent to perform second derivatization treatment, and performing third solid-liquid separation to obtain third liquid materials, namely a rhizoma polygonati sample deriving solution, a polygonatum kingianum sample deriving solution and a polygonatum cyrtonema sample deriving solution; wherein the derivatizing agents comprise N, O-bis (trimethylsilyl) trifluoroacetamide and trimethylchlorosilane;
(4) and performing gas chromatography-mass spectrometry analysis by taking the derived liquid of the polygonatum kingianum sample, the derived liquid of the polygonatum kingianum sample and the derived liquid of the polygonatum cyrtonema sample as samples to be detected, performing multivariate statistical analysis on the obtained data, and identifying three medicinal polygonatum kingianum varieties according to the obtained characteristic metabolites.
The invention respectively grinds and disperses a rhizoma polygonati sample, a polygonatum kingianum sample and a polygonatum cyrtonema sample in methanol to obtain a methanol dispersion liquid of the polygonatum cyrtonema sample, a methanol dispersion liquid of the polygonatum kingianum sample and a methanol dispersion liquid of the polygonatum cyrtonema sample. In the invention, the medicinal sealwort is sealwort, polygonatum kingianum and polygonatum cyrtonema; in the embodiment of the invention, one-to-three-year-old tubers are adopted, and the tubers are quickly frozen by liquid nitrogen and then stored in a low-temperature refrigerator at the temperature of minus 80 ℃ for later use. In the invention, the grinding mode is preferably liquid nitrogen grinding; the powder obtained after grinding is preferably sieved by a 80-mesh sieve, and undersize products are taken for standby.
In the invention, the dosage ratio of rhizoma polygonati to methanol for traditional Chinese medicine is preferably 100 mg: 1.4 mL; the methanol is preferably pre-cooled at-20 ℃ prior to use. In the embodiment of the invention, taking a polygonatum sample as an example, specifically, 100mg of the polygonatum sample with the granularity meeting the requirement is mixed with 1.4mL of methanol and then vortex-oscillated for 1min to obtain a methanol dispersion liquid of the polygonatum sample. In the invention, the preparation modes of the methanol dispersion liquid of the polygonatum kingianum sample and the methanol dispersion liquid of the polygonatum cyrtonema sample are consistent with the preparation mode of the methanol dispersion liquid of the polygonatum cyrtonema sample, and in the subsequent treatment process, if no special description is provided, the treatment modes of the polygonatum kingianum sample and the polygonatum cyrtonema sample are consistent with the treatment mode of the polygonatum cyrtonema sample, and are not repeated.
After a methanol dispersion liquid of a rhizoma polygonati sample, a methanol dispersion liquid of a polygonatum kingianum sample and a methanol dispersion liquid of a polygonatum cyrtonema sample are obtained, the methanol dispersion liquid of the rhizoma polygonati sample, the methanol dispersion liquid of the polygonatum kingianum sample and the methanol dispersion liquid of the polygonatum cyrtonema sample are respectively mixed with a methanol solution of catechol, then ultrasonic treatment and first solid-liquid separation are sequentially carried out, the obtained first liquid materials are respectively extracted in a chloroform-water mixed solvent, and then second solid-liquid separation is carried out to obtain second liquid materials which are respectively an extracting solution of the rhizoma polygonati sample, an extracting solution of the polygonatum kingianum sample and an extracting solution of the polygonatum cyrtonema sample. In the invention, the first liquid materials are three corresponding liquid materials obtained by mixing the methanol dispersion liquid of the polygonatum kingianum sample, the methanol dispersion liquid of the polygonatum kingianum sample and the methanol dispersion liquid of the polygonatum cyrtonema sample with the methanol solution of catechol, performing ultrasonic treatment and performing first solid-liquid separation, and are not mixed liquid of the three liquid materials. In the present invention, the concentration of catechol in the methanol solution of catechol is preferably 0.2 mg/mL; the preferable dosage ratio of the rhizoma polygonati sample to the methanol solution of catechol in the methanol dispersion liquid of the rhizoma polygonati sample is 100 mg: 60 μ L. In the invention, the catechol is used as an internal standard, which is beneficial to the subsequent quantitative detection of metabolites in the medicinal polygonatum. In the embodiment of the invention, taking a polygonatum sample as an example, specifically, a methanol dispersion liquid of the polygonatum sample is mixed with a methanol solution of catechol, vortex oscillation is performed for 1min, and then ultrasonic treatment and first solid-liquid separation are sequentially performed under a sealed condition (for example, a container opening can be sealed by a paraffin film).
In the invention, the temperature of the ultrasonic wave is preferably 40 ℃, and the time is preferably 30 min; the extraction method is preferably vortex shaking for 1 min. In the present invention, the first solid-liquid separation method is preferably centrifugation, and the rotation speed of the centrifugation is preferably 4000rpm and the time is preferably 20 min. The method preferably takes supernatant fluid (namely a first liquid material) obtained after centrifugation, mixes the supernatant fluid with chloroform and water for extraction, and then carries out second solid-liquid separation, wherein the obtained second liquid materials are respectively the extract of a polygonatum sibiricum sample, the extract of a polygonatum kingianum sample and the extract of a polygonatum cyrtonema sample. In the present invention, the amount ratio of the first liquid material to chloroform and water is preferably 1.4 mL: 750 μ L: 1.4 mL; the water is preferably ultra pure water, which is preferably pre-cooled at 4 ℃ prior to use. In the invention, the second solid-liquid separation mode is preferably centrifugation, the rotation speed of the centrifugation is preferably 4000rpm, and the time is preferably 20 min; the supernatant (i.e. the second liquid material) obtained after centrifugation is the extract of the polygonatum sample, the extract of the polygonatum kingianum sample and the extract of the polygonatum cyrtonema sample respectively.
After extracting solutions of a rhizoma polygonati sample, a polygonatum kingianum sample and a polygonatum cyrtonema sample are obtained, removing solvents in the extracting solutions of the rhizoma polygonati sample, the polygonatum kingianum sample and the polygonatum cyrtonema sample, mixing the solutions with a pyridine solution of hydroxylamine hydrochloride respectively for first derivatization treatment, mixing the solutions with a derivatization reagent for second derivatization treatment, and performing third solid-liquid separation to obtain third liquid materials which are respectively a polygonatum kingianum sample derivatization solution, a polygonatum kingianum sample derivatization solution and a polygonatum cyrtonema sample derivatization solution; wherein the derivatizing agents comprise N, O-bis (trimethylsilyl) trifluoroacetamide and trimethylchlorosilane. In the present invention, the concentration of hydroxylamine hydrochloride in the pyridine solution of hydroxylamine hydrochloride is preferably 20mg/mL, and the volume ratio of the pyridine solution of hydroxylamine hydrochloride to the extract solution of the polygonatum sample is preferably 60 μ L: 1 mL. The solvent removing mode is not specially limited, and the nitrogen is adopted for drying. The present invention preferably mixes the residue obtained after removing the solvent with a pyridine solution of hydroxylamine hydrochloride, vortexes under a sealed condition for 30s, and then performs the first derivatization treatment. In the present invention, the temperature of the first derivatization treatment is preferably 60 ℃ and the time is preferably 120 min.
After the first derivatization treatment is finished, the obtained system is mixed with a derivatization reagent for second derivatization treatment, and then third solid-liquid separation is carried out. In the present invention, the mass ratio of N, O-bis (trimethylsilyl) trifluoroacetamide (BSTFA) and Trimethylchlorosilane (TMCS) in the derivatization reagent is preferably 99:1, and the volume ratio of the derivatization reagent to the extract solution of the polygonatum sample is preferably 60 μ L: 1 mL. In the present invention, the second derivatization treatment is preferably performed under sealed conditions; the temperature of the second derivatization treatment is preferably 60 ℃ and the time is preferably 90 min. In the present invention, the third solid-liquid separation mode is preferably centrifugation, the rotation speed of the centrifugation is preferably 12000rpm, and the time is preferably 10 min; the supernatant (i.e. the third liquid material) obtained after centrifugation is the derived liquid of the polygonatum sample, the derived liquid of the polygonatum kingianum sample and the derived liquid of the polygonatum cyrtonema sample respectively.
After the derivative liquid of the polygonatum kingianum sample, the derivative liquid of the polygonatum kingianum sample and the derivative liquid of the polygonatum cyrtonema sample are obtained, the derivative liquid of the polygonatum kingianum sample and the derivative liquid of the polygonatum cyrtonema sample are used as samples to be detected to carry out gas chromatography-mass spectrometry, the obtained data are subjected to multivariate statistical analysis, and the identification of three medicinal polygonatum kingianum varieties is realized according to the obtained characteristic metabolites. In the present invention, when the gas chromatography-mass spectrometry is performed, the operating conditions of the gas chromatography preferably include:
DB-5 quartz capillary column 60m × 250 μm × 0.25 μm; split-flow sample injection with the sample injection amount of 1 mu L and the split-flow ratio of 10: 1; the temperature of a sample inlet is 280 ℃; the ion source temperature is 250 ℃; the interface temperature is 250 ℃; temperature rising procedure: the initial temperature is 40 ℃, the temperature is kept for 5min, the temperature is increased to 280 ℃ at the speed of 8 ℃/min, and the temperature is kept for 5 min; the carrier gas is helium, and the flow rate of the carrier gas is 1 mL/min; the solvent delay time is 14 min; the mass detection range is 33-600 in terms of m/z;
the operating conditions for mass spectrometry preferably include:
an electrospray ionization source, wherein the temperature of an ion source is 230 ℃, the temperature of a quadrupole rod is 150 ℃, the full scanning mode is adopted, and the electron energy is 70 eV; the scanning range of the quadrupole rods is 35-780 in terms of m/z.
In the present invention, after the gas chromatography-mass spectrometry is completed, the method for obtaining the characteristic metabolites of the three varieties of medicinal polygonatum sibiricum by performing multivariate statistical analysis on the obtained data preferably comprises the following steps:
converting original data obtained after gas chromatography-mass spectrometry into CDF format files through GCMSTranslator software, then carrying out primary processing (such as noise filtering, peak matching, baseline calibration, area normalization and the like by using R2.7.2 software so as to remove interference of external factors such as impurity peaks and the like caused by column loss and sample preparation), then editing in EXCEL software, carrying out cluster analysis on the data by combining SPSS19.0 software after the primary processing, and introducing the data into Simca-p14.1 software for multivariate statistical analysis; wherein the multivariate statistical analysis comprises Principal Component Analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA);
screening VIP >1 components by PCA and S-plot of OPLS-DA, and screening components with statistical difference (p <0.05) as differential metabolites by combining t test, wherein the differential metabolites are used as characteristic metabolites for identifying three medicinal sealwort varieties.
In the embodiment of the invention, 13 different metabolites are screened from polygonatum and polygonatum kingianum, wherein the metabolites are respectively N, N-diethylformamide, L-proline, pyrrolidone, leucine, L-isoleucine, phosphoric acid, malic acid, psicose, sorbitol, fructose, glucose, inositol and sucrose, namely the 13 different metabolites are contained in polygonatum and polygonatum kingianum;
screening 19 different metabolites from polygonatum sibiricum and polygonatum cyrtonema, namely tebuthiuron, oxalic acid, L-proline, acetic acid, sulfo group, 1-hydrazide (9CI), alanine, undecane, hydroxylamine, dimethyl malonate, leucine, L-isoleucine, phosphoric acid, 2-piperidinecarboxylic acid, L-pyroglutamic acid, ribose, sorbitol, fructose, inositol, sucrose and 1-glycerol monostearate, namely that the polygonatum sibiricum and polygonatum cyrtonema contain the 19 different metabolites;
17 different metabolites are screened from polygonatum kingianum and polygonatum cyrtonema, and are respectively cyanamide, tebuthiuron, oxalic acid, L-proline, alanine, undecane, hydroxylamine, 1, 3-propanediol, pyrrolidone, phosphoric acid, 2-piperidinecarboxylic acid, L-pyroglutamic acid, ribose, citric acid, sorbitol, inositol and sucrose, namely the 17 different metabolites are contained in polygonatum kingianum and polygonatum cyrtonema.
After the characteristic metabolites of the three medicinal polygonatum varieties are obtained, the identification of the three medicinal polygonatum varieties can be realized according to the characteristic metabolites; furthermore, according to the content of each characteristic metabolite, the quality identification and quality control of the three medicinal polygonatum kingianum can be realized, and the identification of the authenticity and quality of the medicinal polygonatum kingianum sold in the market is facilitated (if the content of the active ingredient is higher, the quality is better).
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
1 Instrument and reagent
1.1 Instrument: agilent7000B-5977C gas-chromatography mass spectrometer.
1.2 reagent: the three medicinal rhizoma Polygonati (rhizoma Polygonati, rhizoma Polygonati Yunnanensis and rhizoma Polygonati Odorati) are one-to-three-year-old tubers, chromatographic grade methanol, chromatographic grade pyridine, and other reagents are analytical reagents, and derivatization reagent and catechol are purchased from Shanghai Allan Biotechnology, Inc.
2 preparation of three medicinal rhizoma Polygonati samples
2.1 extraction of metabolites from three medicinal rhizoma Polygonati samples
Taking three kinds of medicinal rhizoma polygonati tubers (quick-frozen by liquid nitrogen and stored in a low-temperature refrigerator at minus 80 ℃) as experimental materials, grinding the tubers in the liquid nitrogen into powder, sieving the powder by a 80-mesh sieve, and carrying out subsequent treatment on the sieved powder, wherein 6 parts of each medicinal rhizoma polygonati sample are arranged in parallel. Adding 100mg powder into 2mL centrifuge tube, adding 1.4mL methanol (-20 deg.C for precooling), vortex oscillating for 1min, adding 60 μ L catechol methanol solution (catechol concentration is 0.2mg/mL), and vortex oscillating for 1 min; sealing the opening of the centrifugal tube by using a parafilm, and putting the centrifugal tube into an ultrasonic cleaner for ultrasonic treatment at 40 ℃ for 30 min; centrifuging at 4000rpm for 20min after finishing the ultrasonic treatment, putting 1.4mL of supernatant into a 10mL centrifuge tube, adding 750 mu L of chloroform and 1.4mL of ultrapure water (precooling at 4 ℃), performing vortex oscillation for 1min, centrifuging at 4000rpm for 20min, sucking 1mL of upper-layer methanol/water mixed solution, and putting the mixture into a 1.5mL centrifuge tube to respectively obtain extract of three medicinal rhizoma polygonati samples.
2.2 derivatization treatment of metabolites of three medicinal rhizoma Polygonati samples
Blowing the extracting solution in a 1.5mL centrifuge tube by using nitrogen, adding 60 mu L of pyridine solution of hydroxylamine hydrochloride (the concentration of the hydroxylamine hydrochloride is 20mg/mL), sealing by using a paraffin film, carrying out vortex oscillation for 30s, placing in an oven for treating for 120min at the temperature of 60 ℃, then adding 60 mu L of derivatization reagent (consisting of N, O-bis (trimethylsilyl) trifluoroacetamide (BSTFA) and Trimethylchlorosilane (TMCS), specifically 99 wt% BSTFA +1 wt% TMCS), sealing by using the paraffin film, reacting for 90min at the temperature of 60 ℃, centrifuging for 10min at 12000rpm after the reaction is finished, absorbing supernatant liquid as the derivatization liquid of three medicinal rhizoma polygonati samples, and placing in a sample inlet bottle to obtain the sample to be detected.
Detection of metabolites of three medicinal rhizoma polygonati samples
Gas chromatography conditions: DB-5 quartz capillary column 60m × 250 μm × 0.25 μm; split-flow sample injection with the sample injection amount of 1 mu L and the split-flow ratio of 10: 1; the temperature of a sample inlet is 280 ℃; the ion source temperature is 250 ℃; the interface temperature is 250 ℃; temperature rising procedure: the initial temperature is 40 ℃, the temperature is kept for 5min, the temperature is increased to 280 ℃ at the speed of 8 ℃/min, and the temperature is kept for 5 min; the carrier gas is helium, and the flow rate of the carrier gas is 1 mL/min; the solvent delay time is 14min, and the mass detection range (m/z) is 33-600.
Mass spectrum conditions: an electrospray ionization (ESI) source with an ion source temperature of 230 ℃ and a quadrupole temperature of 150 ℃, and a full scan mode with an electron energy of 70 eV; the scanning range (m/z) of the quadrupole is 35-780.
The TIC spectrogram obtained by detecting metabolites of three medicinal rhizoma Polygonati samples is shown in FIG. 1.
4 data processing and analysis
Converting the GC-MS original data obtained in the step 3 into a CDF format file through GCMSTranslator software, then carrying out primary processing (such as noise filtering, peak matching, baseline calibration, area normalization and the like) through R2.7.2 software, so as to remove the interference of external factors such as impurity peaks caused by column loss and sample preparation, then editing in EXCEL software, carrying out cluster analysis on the data through the primary processing by combining SPSS19.0 software, and introducing the data into Simca-p14.1 software for multivariate statistical analysis, including Principal Component Analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA), wherein the result is shown in FIGS. 2-6.
Fig. 2 is a cluster analysis diagram of three medicinal polygonatum rhizome samples, and as can be seen from fig. 2, the three medicinal polygonatum rhizome samples are distinguished into three distinct classes, which indicates that the three medicinal polygonatum rhizome samples can be distinguished in the cluster analysis.
FIG. 3 is a PCA score chart of three samples of medicinal rhizoma Polygonati, wherein 1 represents Polygonatum cyrtonema, 2 represents Polygonatum kingianum, and 3 represents rhizoma Polygonati; FIG. 4 is an S-plot diagram of Polygonatum kingianum and Polygonatum kingianum OPLS-DA, FIG. 5 is an S-plot diagram of Polygonatum kingianum and Polygonatum cyrtonema OPLS-DA, and FIG. 6 is an S-plot diagram of Polygonatum kingianum and Polygonatum cyrtonema OPLS-DA. As can be seen from FIG. 3, Polygonati officinalis rhizoma, Polygonati Yunnan Polygonati officinalis rhizoma and Polygonati officinalis rhizoma exhibit obvious separation tendency, which shows that there are obvious differences in metabolite components of the three medicinal Polygonati rhizoma, and R is2And X is 0.735, which shows that the PCA model is better fitted and has higher prediction accuracy. As can be seen from FIGS. 4 to 6, Polygonatum kingianum (R) of Polygonatum sibiricum Red2Y＝0.998，Q20.963), rhizoma Polygonati vs Polygonatum cyrtonema (R)2Y＝0.998，Q20.981), rhizoma polygonati yunnanensis vs polygonatum cyrtonema (R)2Y＝0.99，Q20.952) of three comparisons2Y and Q2All are close to 1, which shows that the OPLS-DA model has high interpretability. Therefore, the metabonomics can be used for identifying three varieties of medicinal polygonatum.
The VIP >1 fraction was further selected by S-plot of PCA and OPLS-DA and the fractions with statistical differences (p <0.05) were selected as differential metabolites in combination with the t-test. Wherein 13 different metabolites are screened from polygonatum and polygonatum kingianum, 19 different metabolites are screened from polygonatum and polygonatum cyrtonema, 17 different metabolites are screened from polygonatum kingianum and polygonatum cyrtonema (the specific result is shown in table 1), and the different metabolites can be used as characteristic metabolites to realize the identification of three medicinal polygonatum varieties; meanwhile, quality identification and quality control of the three medicinal polygonatum kingianum can be realized according to the content of each characteristic metabolite.
TABLE 1 characteristic metabolites of three medicinal polygonatum sibiricum
Figure BDA0002528732140000101
Figure BDA0002528732140000111
Figure BDA0002528732140000121
In table 1, represents significant differences, 0.01< p < 0.05; represents a very significant difference, p ≦ 0.01.
According to the embodiments, the three medicinal polygonatum kingianum are extracted and derivatized under the conditions, and the nonvolatile metabolites in the medicinal polygonatum kingianum can be detected more comprehensively by using gas chromatography-mass spectrometry, so that the detection accuracy is improved; the three medicinal polygonatum kingianum have high separation degree and good peak shape; the three medicinal polygonatum kingianum are obviously distinguished, and the constructed model has high interpretability and prediction accuracy, so that a quick and accurate method is provided for quality identification and quality control of the three medicinal polygonatum kingianum, and the method is favorable for identifying the authenticity and quality of the medicinal polygonatum kingianum sold in the market.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims (10)

1. A method for identifying three varieties of medicinal polygonatum sibiricum, including polygonatum sibiricum, polygonatum kingianum and polygonatum cyrtonema, comprises the following steps:
(1) grinding the rhizoma polygonati sample, the polygonatum kingianum sample and the polygonatum cyrtonema sample respectively and dispersing the grinded samples in methanol to obtain a methanol dispersion liquid of the rhizoma polygonati sample, a methanol dispersion liquid of the polygonatum kingianum sample and a methanol dispersion liquid of the polygonatum cyrtonema sample;
(2) mixing the methanol dispersion liquid of the polygonatum sibiricum sample, the methanol dispersion liquid of the polygonatum kingianum sample and the methanol dispersion liquid of the polygonatum cyrtonema sample with a methanol solution of catechol respectively, then carrying out ultrasonic treatment and first solid-liquid separation in sequence, extracting the obtained first liquid materials in a chloroform-water mixed solvent respectively, and then carrying out second solid-liquid separation to obtain second liquid materials which are an extracting solution of the polygonatum kingianum sample, an extracting solution of the polygonatum kingianum sample and an extracting solution of the polygonatum cyrtonema sample respectively;
(3) removing the solvent in the rhizoma polygonati sample extracting solution, the polygonatum kingianum sample extracting solution and the polygonatum cyrtonema sample extracting solution, mixing the obtained mixture with a pyridine solution of hydroxylamine hydrochloride respectively to perform first derivatization treatment, then mixing the obtained mixture with a derivatization reagent to perform second derivatization treatment, and performing third solid-liquid separation to obtain third liquid materials, namely a rhizoma polygonati sample deriving solution, a polygonatum kingianum sample deriving solution and a polygonatum cyrtonema sample deriving solution; wherein the derivatizing agents comprise N, O-bis (trimethylsilyl) trifluoroacetamide and trimethylchlorosilane;
(4) and performing gas chromatography-mass spectrometry analysis by taking the derived liquid of the polygonatum kingianum sample, the derived liquid of the polygonatum kingianum sample and the derived liquid of the polygonatum cyrtonema sample as samples to be detected, performing multivariate statistical analysis on the obtained data, and identifying three medicinal polygonatum kingianum varieties according to the obtained characteristic metabolites.
2. The method according to claim 1, wherein the grinding is by liquid nitrogen grinding; and (4) sieving the powder obtained after grinding through a 80-mesh sieve, and taking undersize products for later use.
3. The method according to claim 2, wherein the ratio of rhizoma polygonati to methanol for traditional Chinese medicine is 100 mg: 1.4 mL; the concentration of catechol in the methanol solution of catechol is 0.2 mg/mL; the dosage ratio of the rhizoma polygonati sample to the methanol solution of catechol in the methanol dispersion liquid of the rhizoma polygonati sample is 100 mg: 60 mu L of the solution;
the volume ratio of the first liquid material to chloroform and water in the chloroform-water mixed solvent is 1.4 mL: 750 μ L: 1.4 mL.
4. The method according to claim 3, wherein the ultrasound is at a temperature of 40 ℃ for a time of 30 min; the extraction method is vortex oscillation for 1 min.
5. The method according to claim 4, wherein the concentration of hydroxylamine hydrochloride in the pyridine solution of hydroxylamine hydrochloride is 20mg/mL, and the volume ratio of the pyridine solution of hydroxylamine hydrochloride to the extract solution of the rhizoma polygonati sample is 60 μ L: 1 mL.
6. The method of claim 5, wherein the temperature of the first derivatization process is 60 ℃ and the time is 120 min.
7. The method according to claim 6, wherein the mass ratio of N, O-bis (trimethylsilyl) trifluoroacetamide to trimethylchlorosilane in the derivatization reagent is 99:1, and the volume ratio of the derivatization reagent to the extract solution of the rhizoma Polygonati sample is 60 μ L: 1 mL.
8. The method of claim 7, wherein the temperature of the second derivatization process is 60 ℃ and the time is 90 min.
9. The method of claim 1, wherein the operating conditions of the gas chromatograph comprise, in performing the gas chromatograph-mass spectrometer analysis:
DB-5 quartz capillary column 60m × 250 μm × 0.25 μm; split-flow sample injection with the sample injection amount of 1 mu L and the split-flow ratio of 10: 1; the temperature of a sample inlet is 280 ℃; the ion source temperature is 250 ℃; the interface temperature is 250 ℃; temperature rising procedure: the initial temperature is 40 ℃, the temperature is kept for 5min, the temperature is increased to 280 ℃ at the speed of 8 ℃/min, and the temperature is kept for 5 min; the carrier gas is helium, and the flow rate of the carrier gas is 1 mL/min; the solvent delay time is 14 min; the mass detection range is 33-600 in terms of m/z;
the operating conditions of the mass spectrometer include:
an electrospray ionization source, wherein the temperature of an ion source is 230 ℃, the temperature of a quadrupole rod is 150 ℃, the full scanning mode is adopted, and the electron energy is 70 eV; the scanning range of the quadrupole rods is 35-780 in terms of m/z.
10. The method of claim 1, wherein the multivariate statistical analysis comprises principal component analysis and orthogonal partial least squares discriminant analysis.
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